Stimulation of G-protein coupled receptors (GPCR) is the mechanism of action by drugs of abuse such as cocaine and amphetamine (which exert their effects via dopamine receptors), and morphine (which acts on u-opioid receptors). Activation of these receptors is known to modulate such physiological processes as movement, nociception, and most critically in terms of abuse potential, the reward pathway. However, over stimulation of these receptors by drug action, leads to significant short-term and long-term changes within the dopamine and u-opioid GPCR signaling pathways that are as a result the critical determinants for the development of addiction. One such component of these pathways is the Regulator of G-protein Signaling 9 (RGS9), which has been shown to have a critical role in determining the behavioral response to the administration of these drugs of abuse. RGS9 serves as a negative regulator of dopamine and u-opioid action, and upon administration of drugs such as morphine, RGS9 protein levels increase rapidly and demonstrate lasting changes. This in turn can have a profound impact on the behavioral response elicited. It is the hypothesis of this proposal that alterations in RGS9 protein instability is a key determinant behind the volatility observed in RGS9 levels upon drug administration. The objective of this proposal therefore, is to investigate the molecular mechanisms regulating RGS9 degradation, and how these mechanisms are altered by drug action. Preliminary data suggests that ubiquitination/lysosomal targeting is a key component of RGS9 degradation, leading us to test this hypothesis by evaluating specific lysosome inhibitors and their ability to rescue RGS9 from degradation (Aim #1). Targeting to the lysosome via ubiquitin has been shown to be mediated by K63 poly-ubiquitination, and since RGS9 has been shown by us to be poly-ubiquitinated, we will test our hypothesis that this is through K63 linkage via the use of ubiquitin mutant constructs (Aim #1). Further, mapping of specific lysine residues of RGS9 that are ubiquitinated will be performed via site directed mutagenesis (Aim #2). Lastly, the role of alteration in these RGS9 degradation mechanisms detailed in the first two aims, will be evaluated with the administration of morphine in Aim #3, in an effort to delineate the source of RGS9 volatility that is observed with morphine action. The study as a whole therefore will serve to push our knowledge forward of the mechanisms regulating RGS9, a key component of the fundamental processes of reward and motion. It is only by understanding these mechanistic changes that advancement of treatment options will be possible for reward and motion dysfunctions such as drug addiction and movement disorders (i.e. Parkinson's).